1. Field of the Invention
The present invention relates to certain adsorbents (matrices) for use in affinity chromatography techniques, for example, in the purification of various biologically active molecules such as ligands, proteins, hormones, nucleotides and nucleosides, and more especially, relates to the use of soluble polyfunctional "macromolecular" spacers or "arms" to separate a polysaccharide matrix from the specific ligand or protein to be, e.g., purified. Since "macromolecules" such as the graft copolymer of lysine [backbone] and alanine [side chains] can attach to polysaccharide matrices (for example, cellulose, starch and cross-linked polysaccharide gels such as agarose, Sepharose and Sephadex] at multiple points, the overall chemical stability of the bonding of such polymer to the, e.g., gel, and therefore of other substituents coupled to the polymer, is greatly enhanced.
2. Description of the Prior Art
Affinity chromatography has recently been introduced as a method for purification of biologically active compounds. Cuatrecasas et al, Proc. Matl. Acad. Sci. U.S., 61, 636 (1968). The selective isolation and purification of enzymes and other biologically important macromolecules by "affinity chromatography" exploits the unique biological property of these proteins to bind ligands specifically and reversibly. For example, protein to be purified is passed through a column containing an insoluble polymer or gel to which a specific competitive inhibitor or ligand has been covalently attached. Proteins not exhibiting appreciable affinity for the ligand will pass unretarded through the column, whereas those which recognize the inhibitor will be retarded to an extent related to the affinity constant under the experimental conditions. This method is related in principle to the use of "immunoadsorbents" for the purification of antibodies, which principles have also been applied to the purification of nucleotides, complementary strands of nucleic acids, certain species of transfer RMA, and enzymes by utilization principally of hydrophobic polystyrene or hydrophilic cellulose polymers for the insoluble supporting matrix. Thus, the method depends on the affinity of a protein toward its specific hapten or inhibitor, covalently coupled to an insoluble matrix. In order to use this method successfully, the essential group for interaction with the molecules to be purified must be sufficiently distant from the polymer surface to minimize steric interference. Such a distance can be obtained by introducing a spacer molecule between the solid matrix and the molecule bound thereto. Since the only truly effective method available for binding various molecules to the conventional matrices, e.g., agarose or Sepharose, is by the activation thereof with cyanogen bromide (Axen et al, Nature 214, 1302 (1967); Porath et al, Nature, 215, 1491 (1967)], the spacer must contain a free amino group through which the binding is effected.
Several different approaches of introducing the spacer have been used. One has been suggested by Cuatracasas et al at J. Biol. Chem., 245, 3059, (1970), using amino alkyl-Sepharose as a starting material. The amino derivative was then reacted with different functional groups, to which ligands were coupled and used for protein purification. All of these derivatives have strong ion-exchange properties due to incomplete coupling of the ligand to the amino or carboxyl groups of succinylated derivatives. Alternative approaches have been suggested. Compare Wilchek et al, Israel J. Chem., 8, 172 (1970) and Wilchek, FEBS Letters, 33, 70 (June, 1973).
It has also been recognized that ligands attached to agarose by the CNBr activation reaction [directly or through a spacer arm] are slowly released from the matrix into the buffer medium. Ludens et al, J. Biol. Chem., 247, 7533 (1972); Sica et al, Nature (London) New Biol., 244, 36 (1973). The quantity of ligand released [leakage or bleeding] depends on the temperature and on the specific buffer. Such leakage of the bound ligand from the gel may interfere seriously with affinity chromatographic procedures.
Compare also application, Ser. No. 475,305, filed May 31, 1974, now U.S. Pat. No. 3,947,352, and hereby expressly incorporated by reference.